1. Field of the Invention
The invention relates to biosensors for performing electrochemical analysis to determine concentrations of analytes in liquids.
2. Discussion of the Art
Electrochemical assays for determining the concentrations of enzymes or their substrates in complex mixtures of liquids have been developed. Biosensor strips (i.e., biosensors in the form of strips) are useful in medical research and in external testing. In medical research, biosensor strips can function in an invasive manner (i.e., as probes that come into contact with a body fluid, such as whole blood or subcutaneous fluid). In external testing, biosensor strips can function in a non-invasive manner (i.e., as strips that come into contact with blood withdrawn by a syringe or a lancing device). In particular, biosensor strips for biomedical applications (e.g., whole blood analyses) have been developed for the determination of glucose levels in biological samples. In general, biosensor strips comprise electrochemical cells in which there can be working electrodes, counter electrodes, and reference electrodes. The potential of the working electrode is maintained at a constant value relative to that of the reference electrode.
Conventional electrochemical systems having three electrodes employ (1) a working electrode, (2) a reference electrode, and (3) a counter electrode. The reaction that takes place at the working electrode is the reaction that is required to be monitored and controlled. The functions of the reference and counter electrodes are to ensure that the working electrode actually experiences the desired conditions, i.e. the correct potential intended to be applied. The function of the reference electrode is to measure the potential at the interface of the working electrode and the sample as accurately as possible. In an ideal situation, no current passes through the reference electrode. The function of the counter electrode is to ensure that the correct potential difference between the reference electrode and the working electrode is being applied. The potential difference between the working electrode and the reference electrode is assumed to be the same as the desired potential at the working electrode. If the potential measured at the working electrode is not the potential desired at the working electrode, the potential that is applied between the counter electrode and working electrode is altered accordingly, i.e., the potential is either increased or decreased. The reaction at the counter electrode is also equal and opposite to the charge transfer reaction occurring at the working electrode, i.e., if an oxidation reaction is occurring at the working electrode then a reduction reaction will take place at the counter electrode, thereby allowing the sample to remain electrically neutral.
All commercially available electrochemical biosensor strips for determining the concentration of glucose employ two electrodes. In a two-electrode system, there are (1) a working electrode and (2) a dual-purpose reference/counter electrode. The second electrode is called a dual-purpose reference/counter electrode because this electrode acts as a reference electrode as well as a counter electrode. No current passes through an ideal reference electrode, and such an electrode maintains a steady potential; current does pass through a dual-purpose reference/counter electrode, and thus, the dual-purpose reference/counter electrode does not maintain a steady potential during the measurement. At low currents and/or at short durations of time for measurement, the shift in potential is small enough such that the response at the working electrode is not significantly affected, and hence the dual-purpose reference/counter electrode is designated a dual-purpose reference/counter electrode. The dual-purpose reference/counter electrode continues to carry out the function of a counter electrode; however, in this case, the potential that is applied between the dual-purpose reference/counter electrode and the working electrode cannot be altered to compensate for changes in potential at the working electrode. In other words, while conventional electrochemical measurements require three electrodes, in all commercially available biosensor strips, there are only two electrodes, wherein one of the electrodes performs two functions—the reference function and the counter function.
As indicated previously, a reference electrode provides a reference for the voltage applied at the working electrode. The voltage applied must be sufficient to oxidize or reduce the species (molecule or ion) of interest at the surface of the working electrode. The voltage required is determined by the ease of removing or adding an electron to the species of interest. Because this voltage is applied externally (by means of a potentiostat or battery), the reference point should be maintained at a constant value. If the value of the reference point changes with time, the external voltage applied should be varied accordingly. Commercially available biosensor strips are very sensitive to the quality of the electrode that performs two functions (the reference function and the counter function). If that electrode is of poor quality, the voltage applied at the working electrode (by means of a battery or potentiostat) will not be maintained at a constant value, resulting in variation in the response of the biosensor strip from sample to sample. This variation depends on the hematocrit (which affects solution resistance) and concentration of the analyte (which affects current). In most electrochemical measurements, the current is measured at a constant applied voltage.
In a biosensor strip, the electrodes are separated from each other. The space between the electrodes results in the loss of voltage control at the working electrode. The voltage experienced at the working electrode therefore is lower than that applied. The difference between the voltage applied and the voltage experienced at the working electrode is a product of the current passing between the dual-purpose reference/counter electrode and the working electrode and the resistance of the solution. Also, on account of the current passing through the circuit, the dual-purpose reference/counter electrode becomes polarized. In other words, the flow of current through the dual-purpose reference/counter electrode brings about a reduction reaction at the electrode, thereby changing the chemical composition of the dual-purpose reference/counter electrode. This change in chemical composition brings about a change in the potential at the dual-purpose reference/counter electrode, and hence a change in the voltage applied.
A biosensor strip having three electrodes would be preferred in any electrochemical measurement that involves the application of an external voltage and measurement of current. However, due to constraints of sample volume (lower volume requirements), all electrochemical biosensor strips commercially available use only two electrodes. Precise control of the voltage difference between the working electrode and the reference electrode must be maintained, but such control is difficult to achieve in a two-electrode system.